Traditional genetics depended upon mutations or pre-existing genetic polymorphisms which were discovered in a species. The only experimental approach to widen the scope of genetic variants available for study was mutagenesis followed by specific screening or fortuitous recovery of relevant alleles. Animal breeding depends upon selection from suitable variation either in or introduced into the stock. The major tool for genetic analysis was breeding segregation studies and direct phenotypic analysis.
Notwithstanding the great theoretical and practical interest in mammalian genetics the conventional genetic analysis of experimental mammals, because of their relatively small litter sizes and long life cycles, has been handicapped vis-a-vis other experimental animal systems. Mammalian genetics has, however, benefitted from the study of human genetics where although there is no experimental breeding, detailed observation of a very large population has allowed investigation both of polymorphisms and of very rare mutations, and statistical methods for analysis of data available from human pedigrees of genetic segregation have become highly refined.
Added to this, methods for non-meiotic genetic analysisxe2x80x94somatic cell genetics and more recently direct molecular-biological analysis, have been and indeed are being very effectively applied as well as being combined with pedigree analysis so that mammalian genetic maps and knowledge of gene sequence data are advancing rapidly.
This is, however, an observational analysis. Our understanding of genetic function and also, in experimental animals, the practical application of our genetic knowledge requires the ability deliberately to modify the genome, preferably in a manner not entirely reliant upon screening the accidents of nature. Thus the concept of a reverse mammalian genetics emerges where the effect of specific genetic modification may be studied in the context of the intact organism.
Genes of interest are now not only being identified from the results of the intensifying mammalian genetic analysis but importantly also through their molecular biology and by cross homology to those of other species. In a great number of cases there are genes which have been identified in mice through their molecular biology, by analogy with those of other species (e.g. human disease syndromes or Drosophila genetics) or from the biochemistry of their protein products but for which there is no lack-of-function allele and thus no rigorous genetic test of function. Neither are these mutations of protein structure or of genetic control. These can only be provided by creating such alleles. In the field of practical application to domestic farm animals, potential alteration of normal physiology which may be desirable, deletion or modification of function of controlling genes may be just as important as overexpression of others. The technology to undertake such targetted gene deletion or modification has now reached feasibility as exemplified by creation of null alleles at the hypoxanthine phosphorbosyl transferase genexe2x80x94HPRTxe2x80x94locus [1] [2]. The development of methods which will allow targetting and screening for deletion of function or specific modification of any gene whose sequence is known is now well underway and this is clearly a realistic proposal. It will become an available routine technique for mouse cells in the next year or so and with the development of domestic animal embryonic stem cells will be immediately applicable to these species and may well become the transgenic route of choice.
Transgenic animals possess an alteration in their DNA which has been stably incorporated into the genome as a result of intentional experimental intervention. Typically this results from the additional exogenous foreign DNA or novel DNA constructs. With the advent of specific gene targetting we should not necessarily exclude from the definition of transgenesis specific modification of endogenous gene sequences by direct experimental manipulation.
A fully experimental approach to mammalian genetics is very rapidly becoming a reality through the use both of conventional zygote injection transgenics and of embryonic stem cells. The latter approach allows extensive in vitro genetic manipulation, selection and screening prior to whole animal reconstruction. Thus both an experimental molecular genetics and the ability to design genetic changes in animals are available. For practical purposes the mouse has been the species of choice for such studies but it is important to be able to extend the methods developed in the mouse to larger domestic farm animal species with the intention of their practical application. The new experimental mammalian genetics allows for testing of genetic modifications in vivo and designing genetic modification of a target species. One of the most important prospects is the construction of experimental animal models of disease for pharmaceutical testing and developments. The other is for specific modification of domestic farm animals to create more desirable qualities for food production, disease resistance, and biopharmaceutical protein production.
Although DNA micro-injection is the most commonly used method of generating transgenic animals, alternatives include embryo infection using recombinent retroviral vectors incorporating the transgene and also the use of pluripotential embryonic stem (ES) cells.
In order to introduce genetic alterations into a mammal it is necessary to transform genetically a cell the progeny of which can give rise to all or to the desired part of the intact organism. zygote micro-injection [4] achieves transgenesis by transformation of the embryo""s genome at the single cell or very early cleavage stage. As the germ line in mammals is segregated from somatic progenitor lineages at the early primitive-streak stage transformation of embryonic cells before this stage for instance by retroviral vector infection of cleavage embryos [5] may provide animals which are transgenic both somatically and in the germ line. Genetic transformation of cells after this stage will lead to either germ line or somatic genetic mosaicism. Where stem cells may be isolated from the organism these may be transformed and used to re-colonise their target tissue and the use of such techniques is exemplified by haematopoetic stem cell manipulation e.g. [7]. This type of approach to somatic transgenesis is likely to be the only ethical route for human gene therapy and could well prove particularly useful for genetic modification of domestic livestock and when the stem cells may be maintained in tissue culture prior to their use to reconstitute their target tissue there is the advantage that selection for the desired transformants may preceed reconstitution. See for example Edwards"" use of transient culture of mammary epithelial stem cells [8]. Cells isolated from an embryo before segregation of the germ line are able to provide a genetic vehicle for germ line transgenesis. Whereas embryonic stem cells have been isolated from mice [9] [10] and cells which seem likely to have such properties from hamster, [11] it is by no means apparent that cells of a similar type may be necessarily isolated from other non-rodent embryos. Moreover it is unlikely that the methods as described for mouse and utilised for hamster will be directly applicable to other embryos. Indeed the reported failure (notwithstanding the optimistic title) of some competent researchers in the field [12] to isolate sheep embryonic stem cells by a method based upon that used for mouse embryonic stem cells indicates this. Others have isolated cells but failed to maintain lines or demonstrate their pluripotentiality [15]. Past failures may have been due to the expectation that the cells would be fast-growing and resemble those of the mouse. It was indeed reported that malignant transformation was necessary in order to overcome the inherent quiescence of the embryonic disc [6].
There have also been numerous attempts which have been orally reported at various scientific meetings which have been unsuccessful. When the mouse embryonic stem cells were first isolated virtually every expected property was predicted and the embryonic stage at which they might be found was clearly identified [13]. None of this background is available for putative ungulate embryonic stem cells.
We have discovered that the methods which have been established and described for the isolation of embryonic stem cells from mouse embryos and successfully applied to hamster embryos are NOT applicable to ungulate embryos as exemplified by bovine and porcine embryos. In particular the most important step in embryonic stem-cell isolationxe2x80x94identification and isolation of the stem cells from other cell types is quite differently based as is the necessary tissue-culture handling of the cells.
In accordance with the present invention, there is disclosed a method of selecting and growing pluripotential embryonic stem cells isolated from an ungulate species blastocysts of embryos that develop by way of an embryonic disc, comprising growing blastocysts in tissue culture growth medium which includes both heat-inactivated new born calf serum and heat-inactivated fetal calf serum; disaggregating the blastocysts either after spontaneous hatching or after mechanical removal of the zone pellucida; growing stem cell colonies from the disaggregated cells in tissue culture growth medium; selecting stem cell colonies by morphological characteristics; and growing the selected stem cells in tissue culture growth medium, wherein the morphologically selected cells are capable of culture in a tissue culture dish to exhibit the following morphological features:
a) they are round cells, tightly packed with large nuclei in relation to cytoplasm, and fairly prominent nucleoli;
b) they grow in tightly adherent colonies, and as the colonies get larger the cells tend to flatten out in the center of the colony, with the colony having an outer rim of cells of the form described in a), and
c) on trypsinization of such a colony it may be seen that the outer, less flattened cells of a larger colony or all the cells of a smaller colony without central flattening are readily disaggregated by trypsinization into small spherical cells which have a bright phase contrast appearance, and if observed after a short time of incubation at 37xc2x0 C. show lobular pseudopodia.